It is estimated that sugar chains are involved not only in life phenomena such as development, differentiation and cell recognition but also in occurrence and progress of inflammations, cancers, infections, auto-immune diseases and a number of other diseases [A. Kobata, S. Hakomori and K. Nagai: Glycobiology Series (1) to (6), Kodansha (1993), Glycobiology, 3, 97 (1993)].
Sugar chains exist not only as glycoproteins, proteoglycans or glycolipids, in which they are added to proteins or lipids, but also as oligosaccharides.
Sugar chains having Galβ1-3GlcNAc structure are called type 1 sugar chains, and constitute core sugar chains of Lewis blood type antigens and cancer-related sugar chain antigens. The Lewis blood type antigens include not only Lewis a sugar chain [Galβ1-3(Fucα1-4)GlcNAc] but also Lewis b sugar chain [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc]. Sialyl-Lewis a sugar chain [NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc] and sialyl-Lewis c sugar chain (NeuAcα2-3Galβ1-3GlcNAc) are cancer-related sugar chains detected highly frequently in cancers in mainly the digestive system such as colon cancers, pancreatic cancers, etc., and antibodies against sialyl-Lewis a sugar chain and sialyl-Lewis c sugar chain are utilized for serodiagnosis of cancers.
It is revealed that adhesion of adhesion molecule selectins (E-, P- and L-selectins) and their sugar chain ligands (sialyl-Lewis x sugar chains or their related sugar chains) is involved in accumulation of leukocytes in inflammatory sites and homing of lymphocytes to lymph nodes.
Because sialyl-Lewis a sugar chain, i.e. structural isomer of sialyl-Lewis x sugar chain [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc] binds to selectins, sialyl-Lewis a sugar chain is considered to participate in cancer metastasis. Further, it is reported that the expression level of sialyl-Lewis a sugar chain in colon cancers and pancreatic cancers is correlated with poor prognosis of cancers.
The Galβ1-3GlcNAc structure is synthesized by a GlcNAc β1,3-galactosyltransferase. To date, genes of three GlcNAc β1,3-galactosyltransferases (β3Gal-T1, β3Gal-T2, β3Gal-T3) have been cloned, and the acceptor substrate specificity of each enzyme has been analyzed [Japanese Published Unexamined Patent Application No. 181759/94, J. Biol. Chem., 273, 58–65 (1998), J. Biol. Chem., 273, 433–440 (1998), J. Biol. Chem., 273, 12770–12778 (1998)]. Further, another β1,3-galactosyltransferase (β3Gal-T4) having different substrate specificity has been cloned [J. Biol. Chem., 272, 24794–24799 (1997), J. Biol. Chem., 273, 12770–12778 (1998)]. β3Gal-T4 synthesizes ganglioside GA1, GM1 or GD1b, but not the Galβ1-3GlcNAc structure.
If GlcNAc β1,3-galactosyltransferase involved in the synthesis of sialyl-Lewis a sugar chain and sialyl-Lewis c sugar chain as cancer-related sugar chains can be identified in cancers in the digestive system such as colon cancers, pancreatic cancers, etc., more accurate diagnosis of cancers would be feasible by examining said enzyme or the expression level of a gene of said enzyme. Further, it is expectable that cancer metastasis could be inhibited by regulating the activity of said enzyme or the transcription and translation of said enzyme gene. However, neither said enzyme nor said enzyme gene has been identified. A GlcNAc 1,3-galactosyltransferase has been partially purified from colon cancer cell line Colo205, but nobody has achieved isolation of said enzyme, determination of the amino acid sequence of said enzyme, or isolation of a gene of said enzyme [J. Biol. Chem., 262, 15649–15658 (1987), Archi. Biochem. Biophys. 270, 630–646 (1989), Archi. Biochem. Biophys. 274, 14–25 (1989)].
A sugar chain having an ability to bind to selectins is useful as a selectin antagonist to treat and prevent inflammations and cancer metastasis. Accordingly, it is estimated that β1,3-galactosyltransferases involved in the synthesis of sialyl-Lewis a sugar chains in cancers in digestive system such as colon cancers and pancreatic cancers is also applicable to efficient synthesis of selectin antagonists.
It is known that various oligosaccharides occur in human milk [Acta Paediatrica, 82, 903 (1993)]. Lacto-N-tetraose (Galβ1-3GlcNAcβ1-3GlcNAcβ1-4Glc) is contained in human milk and estimated to prevent infants from being infected with viruses or microorganisms. Further, lacto-N-tetraose has the activity of promoting the growth of bifidobacterium as benign enteric bacterium. On the other hand, there are few types of oligosaccharides occurring in milk from animals such as cattle, mice, etc., and a majority thereof are lactose, and trisaccharides or higher oligosaccharides scarcely occur [Acta Paediatrica, 82, 903 (1993), J. Biol. Chem., 270, 29515 (1995)].
It would be considered significantly advantageous in industry if we could produce efficiently various oligosaccharides having lacto-N-tetraose as a backbone. It is therefore an industrially important task to develop an enzyme having a higher activity of synthesizing lacto-N-tetraose than the activity of GlcNAc β1,3-galactosyltransferase so far cloned.